Molecular mechanisms underlying cell death are a major focus of current biomedical research as aberrant cell death is involved in the pathogenesis of a vast number of diseases including the CNS disorders. Cell death can be broadly separated into two extreme categories termed apoptosis and necrosis. Apoptosis, also referred to as programmed cell death, is a process in which a cell dies by activation of an intrinsic genetic program. Apoptosis plays an important role in the development and aging processes in the central nervous system (CNS). Abnormality in apoptosis has been linked to pathogenesis of neurodegenerative diseases and implicated in neuropsychiatric disorders. We have used primary cultures of rat CNS neurons and neurally related cell lines as a model to study molecular mechanisms underlying neuronal apoptosis. We found that glyceraldehyde-3-phospahate dehydrogenase (GAPDH), a glycolytic enzyme, is over- expressed during apoptosis induced by aging of rat cerebellar granule cells in culture. Antisense oligonucleotides to GAPDH block GAPDH over-expression and effectively delay age-induced apoptosis of these cerebellar neurons. These results provide the first evidence for a role of GAPDH in neuronal apoptosis. By the same criteria, we found that GAPDH is involved in age-induced apoptosis of rat cerebral cortical neurons and apoptosis of cerebellar granule cells induced by extracellular potassium deprivation and exposure to cytosine arabinoside (AraC). In the case of AraC-induced apoptosis, we found that the cell death is robustly protected by the neurotrophins BDNF and NT 4/ 5, but not NT-3, and by inhibitors of phosphatidylinositol 3-kinase. The apoptosis is also associated with an increased expression of two death genes, p53 and Bax, preceding the over-expression of GAPDH. An antisense oligonucleotide to p53 reduces GAPDH induction and protects against AraC-induced apoptosis, suggesting that GAPDH over-expression depends on p53 expression. Moreover, tranfection of the p53 gene into PC12 pheochromocytoma cells induces GAPDH over-expression and causes ultimate cell death, suggesting that GAPDH is a novel target gene regulated by p53. AraC-induced GAPDH over-expression is predominantly accumulated in the nucleus assessed by subcellular fractionation study and electromicroscopic immunohistochemistry. Translocation of GAPDH to the nucleus occurs in parallel with a loss of GAPDH glycolytic and uracil glycosylase activities, suggesting an alteration in the structure and function of nuclear GAPDH. We also demonstrated that at least six GAPDH isoforms can be detected in the nucleus of cerebellar granule cells. These nuclear isoforms differ in their abundance and are translocated to the nucleus according to a distinct time-course following AraC treatment. Evidence is also available that nuclear GAPDH accumulation precedes activation of caspase-3 and cleavage of its nuclear substrate lamin B1. Using hypothalamic GT1-7 cells, we found that GAPDH over-expression and nuclear translocation induced by thapsigargin is markedly inhibited by over-expression of Bcl-2, a major cytoprotective protein. Recently, GAPDH has been shown to bind specifically to gene products of degenerative diseases such as Alzheimer's disease, Huntington's disease, DRPLA and spinocerebellar ataxia. We have studied GAPDH abnormalities in a transgenic mouse model of Huntington's disease in which the disease protein (huntingtin) expresses expanded (89) CAG repeats. We found that GAPDH is overexpressed in neurons of discrete brain areas such as the hippocampal formation, caudate-putamen and globus pallidus, compared with wild type control. Confocal microscopic analysis of intracellular GAPDH localization revealed a predominant increase in the nucleus. Thus, mutation of huntingtin is associated with GAPDH overexpression and nuclear translocation in discrete populations of neurons in selective brain areas. Our results suggest that over-expression of GAPDH in CNS neurons could induce neuronal apoptosis and ultimate neurodegeneration. Moreover, the ability of GAPDH antisense oligonucleotides to rescue neurons from undergoing apoptotic cell death also suggests that such oligonucleotides may be useful for treating neurodegenerative diseases. We also found that ONO-1603, a potential anti-dementia drug, protects against age-induced apoptotic death of cerebral cortical neurons in a nanomolar concentration range. Additionally, ONO-1603 and tetrahydroaminoacridine, a FDA-approved anti-dementia drug, protects cerebellar neurons against low KCl- induced apoptosis. Thus, apoptotic death of CNS neurons in culture could be a valuable tool for screening more effective drugs in the treatment of dementia associated with Alzheimer's disease. In an attempt to further elucidate molecular mechanisms underlying apoptosis, we have used cerebellar granule cells treated with AIDS-related neurotoxins, such as 3-OH- kynurenine (3-HK) and quinolinic acid, which are tryptophan metabolites and whose levels are elevated in the brain of AIDS patients. We found that 3-HK induces apoptosis in the range of 50-1000 micro-M, while quinolinic acid is ineffective. The 3-HK neurotoxicity is potentiated by superoxide dismutase (SOD) and a cell-permeable SOD mimetic, MnTBAP. Both 3-HK-induced and SOD-potentiated neurotoxicities are blocked by catalase, suggestive of hydrogen peroxide formation. We also found that 3-HK induces apoptotic death of PC12 pheochromocytoma cells and hypothalamic GT1-7 cells. In both cell types, 3-HK-induced apoptosis is robustly protected by dantrolene, a drug that inhibits Ca-2+ efflux from the endoplasmic reticulum to the mitochondria. Moreover, overexpression of Bcl-2, an anti-apoptotic gene product, in GT1-7 cells arrests 3-HK-induced neurotoxicity. Thus, pharmacological manipulation with dantrolene and/or gene therapy with Bcl-2 are potentially useful for the treatment of 3-HK-related neurodegeneration and other forms of neurodegenerative diseases. Glutamate excitotoxicity has been implicated in a variety of neurodegenerative diseases. Using cerebellar granule cells as a model system, we have studied mechanisms underlying glutamate-induced apoptosis in these cell types. We found that glutamate excitotoxicity is associated with increased expression of apoptotic proteins, p53 and Bax, but with decreased expression of the cytoprotective protein, Bcl-2. The excitotoxicity is also concurrent with inhibition of the cell survival factors, Akt-1 and p-CREB (cyclic AMP-responsive element binding protein) due to activation of protein phosphatase PP2A and PP1, respectively. Additionally, glutamate-induced apoptosis is preceded by activation of p38 kinase and JNK (c-Jun N-terminal kinase), and suppression of these kinase activities results in neuroprotection. Activation of these kinases results in phosphorylation and activation of p53. Moreove, glutamate excitotoxicity mediated through NMDA receptors involves nuclear accumulation of GAPDH. Long-term treatment with valproate, an anticonvulsant and mood stabilizer, normalizes this GAPDH nuclear translocation and protects against excitotoxicity. We also showed that GAPDH interacts with histones in nuclei of neurons and this interaction is weakened by histone acetylation. Synergistic neuroprotective effects of valproate and lithium, another mood stabilizer, have been obtained. In an attempt to elucidate the role of numerous proteins in apoptosis and neuroprotection, we have succeeded in developing high transfection efficiency technology of siRNA in primary cultures of neurons.